The main focus of this proposal will be on the design and testing of oncolytic vaccinia vectors expressing STAT3-silencing peptides such as PIAS3 in order to directly enhance the oncolytic and immunotherapeutic activity of the vectors, to add additional therapeutic mechanism and to sensitize tumors to other therapies. The transcription factor STAT3 (signal transducer and activator of transcription 3) is strongly associated with a poor prognosis in multiple cancer types. Activated STAT3 promotes cell growth and prevents apoptosis in transformed cells, maintains an immunosuppressive state in tumor-resident leukocytes and has been associated with CSC differentiation and EMT. Thus, targeting of STAT3 has tremendous therapeutic potential. As such, inhibition of STAT3 has been shown to trigger apoptosis, promote anti-tumor immunity, sensitize tumors to further therapies, and to enhance immunogenicity to cancer vaccines. Oncolytic viruses are live viral cancer therapies that have their replication restricted to malignant cells. Interest in this platform has recently sen a resurgence due to success in several randomized clinical trials. Multiple oncolytic vaccinia virus (VACV) vectors have entered clinical testing, including one expressing GM-CSF as an immunotherapeutic transgene that recently reached its primary endpoints in randomized Phase IIb clinical testing. This highlights the potential of these vectors to act as immunotherapies and supports the hypothesis for combining STAT3-silencing with oncolytic VACV therapy to create potent cancer treatments. We have demonstrated that STAT3 inhibition can enhance viral-induced cell killing in a panel of renal and pancreatic cancer cell lines. It is therefore believed that vaccinia expressing human PIAS3 (protein inhibitor of activated STAT3), which is known to block STAT3 from binding to DNA and thus inhibit its function as a transcription factor, would act as a powerful cancer therapeutic. This was supported by the observation that cells transfected to express functional domains of PIAS3 were sensitized to virus-induced cell death and simultaneously increased VACV replication. Our initial data has also indicated that inhibition of STAT3 could sensitize tumor-associated macrophages (TAMs) to viral replication, so also enhancing the immunotherapeutic activity of this combination. Our preliminary in vitro data therefore strongly suggest a therapeutic enhancement to both VACV oncolytic and immunotherapeutic activity through combination with STAT3 inhibition. Here we will design and test oncolytic vaccinia vectors expressing PIAS3 or other STAT3-silencing peptides. The effectiveness of these novel therapeutic vectors will be examined using human tumor cell lines and in vivo in mouse tumor models. It is believed that vectors produced here would not only provide therapeutic advantages over the current clinical vectors but could also synergize with other therapies to further increase their tumor-killing potential.